1. Field of the Invention
The present invention relates generally to the fields of molecular biology, immunology and oncology. More particularly, it concerns the development of monoclonal antibodies and fragments thereof and their use in the prevention and therapy of diseases associated with abnormal vascular proliferation, such as cancer.
2. Description of Related Art
Tumor markers are substances produced by tumor cells or by other cells of the body in response to cancer. These substances can be found in the blood, in the urine, in the tumor tissue, or in other tissues, such as the surrounding tissues including the vasculature that feeds the tumor. Different tumor markers are found in different types of cancer, and levels of the same tumor marker can be altered in more than one type of cancer. In addition, tumor marker levels are not altered in all people with cancer, especially if the cancer is early stage. Some tumor marker levels can also be altered in patients with non-cancerous conditions. As such, tumor markers are highly useful in diagnostic procedures, and in certain cases, can even be used as therapeutic targets, i.e., to allow a therapy to discriminate between diseased and healthy tissues.
One successful example of a tumor marker that has been used to target a cancer therapy is the proto-oncogene human epidermal growth factor receptor 2 (HER-2; also known as neu and ErbB-2). Epidermal growth factor receptors are proteins embedded in the cell membrane that help regulate cell growth, survival, adhesion, migration, and differentiation. These functions are amplified in some cancers, notably some breast cancers, in which there is an amplification of the HER-2 gene or over-expression of its protein product, which causes breast cells to reproduce uncontrollably. Antibodies to the protein HER-2, such as HERCEPTIN, are currently used to treat breast cancer.
Another way of exploiting tumor markers is to attack molecules with which the marker interacts. For example, vascular endothelial growth factor receptor (VEGFR) is often up-regulated in vascular tissue surrounding tumors, and tumor cells can secrete excess VEGF. As a result, blood vessel growth proceeds in an uncontrolled fashion, much like the growth of the tumor. The anti-VEGF antibody, Bevacizumab, binds to VEGF and prevents it from interacting with its cognate receptor. This in turn results in impaired blood vessel development surrounding the tumor, thereby limiting tumor growth. However, just as with many other cancer therapies, not all cancers and not all patients will respond to these kinds of treatments. One of the limitations of current tumor marker strategies in cancer therapy is that these therapies cannot separate physiological and pathological angiogenesis. Consequently, various effects associated with the use of these agents have been reported (Higa and Abraham, 2009).
Tumor endothelial Marker 8 (TEM8), an 85 kDa integrin-like cell surface receptor, was originally identified as one of several unrelated genes (called TEM1-TEM9) overexpressed in vascular endothelial cells derived from tumor versus normal colorectal tissues (St Croix et al., 2000). Subsequent studies have shown that TEM8 is overexpressed in the blood vessels of a variety of human cancer types (St Croix et al., 2000; Nanda et al., 2004).
Insights into the physiological functions of TEM8 are beginning to emerge. In vitro studies suggest that TEM8 can bind collagens, such as collagen I and collagen VI which, in turn, can promote the migration of endothelial cells (Nanda et al., 2004; Hotchkiss et al., 2005). Migration of cells on extracellular matrix is dependent on actin cytoskeleton reorganization, and recent studies suggest that the TEM8 cytosolic domain may link extracellular matrix molecules to the actin cytoskeleton (Werner et al., 2006; Go et al., 2009). However, it is unclear which components of the actin cytoskeleton are involved in binding TEM8 under physiological conditions, and how this binding contributes to TEM8 function. TEM8 may also be involved in collagen uptake through an endocytosis-mediated degradation pathway, as TEM8 knockout mice are viable but display an excess buildup of collagen in select organs (Cullen et al., 2009).
TEM8 shares 58% amino acid identity with CMG2, another cell surface receptor that binds extracellular matrix (ECM) proteins, in this case laminin and collagen type IV (Bell et al., 2001). Both TEM8 and CMG2 share an integrin-like von Willebrand factor A domain in their extracellular region. TEM8 and CMG2 have both been found to bind anthrax toxin proteins (Bradley et al., 2001; Scobie et al., 2003), and have therefore been given the alternative names anthrax toxin receptor 1 (ANTXR1) and ANTXR2, respectively. Protective antigen (PA) is the subunit of anthrax toxin responsible for binding TEM8 or CMG2, and the PA-receptor interaction is critical for toxin entry into cells. TEM8 is highly conserved, and mouse TEM8 protein, which shares 98% amino acid identity with human TEM8, is also overexpressed in mouse tumor vessels (Carson-Walter et al., 2001).
TEM8 is unique among the original TEMs identified in that it has not been found to be detected in the angiogenic vessels of adult ovaries, and in TEM8−/− knockout mice developmental angiogenesis appeared unaffected (St Croix et al., 2000; Nanda et al., 2004; Cullen et al., 2009). However, in tumor challenge studies tumor growth was impaired in TEM8 knockout compared to wild-type mice (Cullen et al., 2009).